T Cells are one of cell populations which play a major central role in immune system as a biological defense system against various pathogens. Such T cells are roughly classified into CD4-positive helper T cells and CD8-positive cytotoxic T cells. The former relates to the promotion of immune response and the latter relates to the exclusion of virus-infected-cells and tumor cells. Helper T cells are further classified into both T helper type 1 cells which promote cell-mediated immunity and T helper type 2 cells which promote humoral immunity. These T cells with such different properties have functions of excluding pathogens and of acquiring infection tolerance under well-balanced immune response.
Usually, under the normal immune response, no exclusion system functions because immunological tolerance against self-antigens that constitute living bodies has already been established. Self-reactive T cells induce either cell death or non-reaction against self-antigens. In particular, it has been said that regulatory T cells positively relate to such regulation. Such regulatory T cells are defined as those which have function of suppressing other T cells and, recently, they have been researched as a cell population with function of suppressing a particular immune response, reporting that there exist different types of regulatory T cells with different cell-surface markers, producing cytokines, immunosuppressive mechanisms, etc.
Among these regulatory T cells, most well-studied are, for example, CD4-positive CD25-positive regulatory T cells (CD4+CD25+ Treg cells), disclosed by Sakaguchi in “Annual review of immunology”, Vol. 22, pp. 531-562, 2004. In the past results in many researches, since the above cells exhibit CD4-positive CD25-positive features, CD4 and CD25 are recognized to be used as markers for regulatory T cells. As for CD4-positive CD25-positive regulatory T cells, Fontenot et al., “Nature immunology”, Vol. 4, No. 4, pp. 330-336, 2003, discloses “FOXP3”, a transcription factor; and Takahashi et al., “Journal of experimental medicine”, Vol. 192, No. 2, pp. 303-309, 2000, discloses “CTLA-4”, a cytolytic T lymphocyte associated antigen-4, which induces T cells into unreactive conditions. Cosmi et al., “Blood”, Vol. 102, No. 12, pp. 4107-4114, 2003, discloses a T cell having a CD8-positive CD25-positive marker and immunosuppressive activity.
As described above, CD8-positive cytotoxic T cells are well known as T cells having cytotoxic activity, which show a relatively high specific, cytotoxic activity against target cells. Japanese Patent Kokai No. 2005-245430 discloses that CD8-positive cytotoxic T cells exert a specific, cytotoxic activity against tumor cells when co-cultured with them or a protein characteristic to them.
International Patent Publication No. WO 2007/105797 A1 applied for by the same applicant as the present invention discloses that there can be obtained a novel human T cell population, which exerts cytotoxic and immunosuppressive activities against activated human T cells when mononuclear cells collected from human blood are co-cultured with stromal cells. The human T cell population is expected for use in researches and medicals because substantially most of the cells in the T cell population are positive for cell-surface-antigens CD3, CD25, and CD28 and for T cell antigen receptor α/β; contain all three groups of a CD4-positive CD8-positive T cell group, CD4-positive CD8-dimly positive T cell group, and CD4-negative CD8-positive group; and have both cytotoxic and immunosuppressive activities.
The method for producing a T cell population, which has both cytotoxic and immunosuppressive activities disclosed in International Patent Publication No. WO 2007/105797 A1 (hereinafter, the method is described as “conventional method” throughout the specification), is a simple method containing the steps of continuously co-culturing mononuclear cells collected from a fresh umbilical cord blood or peripheral blood in the absence of IL-2, co-culturing the generated blast cells with stromal cells in the presence of IL-2, and further proliferating the resulting cells to form a desired T cell human population which has both cytotoxic and immunosuppressive activities. The conventional method, however, requires mononuclear cells derived from a fresh human umbilical cord blood or peripheral blood and needs a long period of time, i.e., three to four weeks, to induce and proliferate blast cells. In practicing the conventional method, the following drawbacks are still remained; a fresh umbilical cord blood or peripheral blood is inevitable, a long culturing period of about four weeks is needed, and a possible percentage of inducing a desired human T cell population is usually low, i.e., about 10% (one out of ten populations). Any industrially applicable method for producing the same has not been established yet. Under these circumstances, there required is the establishment of a method for efficiently producing a desired human T cell population, which has both cytotoxic and immunosuppressive activities, disclosed in International Patent Publication No. WO 2007/105797 A1.